kQ-D 1990 Oxford University Press

Nucleic Acids Research, Vol. 18, No. 23 7055

Structural analysis of the 5' domain of the HeLa 185 ribosomal RNA by chemical and enzymatic probing Valsan Mandiyan* and Miloslav Boublik Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110, USA Received July 24, 1990; Revised and Accepted October 31, 1990

ABSTRACT The secondary structure of HeLa 18S rRNA was investigated by a combination of chemical and enzymatic probing techniques. Using four chemical reagents (DMS*, kethoxal, DEPC and CMCT) which react specifically with unpaired bases and two nucleases (RNase Ti and cobra venom nuclease) which cleave the ribopolynucleotides at unpaired guanines and helical segments, we have analyzed the secondary structure of the 5' domain of 18S rRNA isolated from HeLa 40S ribosomal subunits. The sites at which chemical modifications and nuclease cleavages occurred were identified by primer extension using synthetic deoxyoligonucleotides and reverse transcriptase. These studies led to the deduction of an intra-RNA pairing pattern from the available secondary structure models based on comparative sequence analysis. Apart from the general canonical pairing we have identified noncanonical U-U, G-A, A-G, A-C, C-A and G-G pairing in HeLa 18S rRNA. The differential reactivity of bases to chemical reagents has enabled us to predict the possible configuration of these bases in some of the noncanonical pairing. The abscence of chemical reactivities and cobra venom nuclease sensitivity in the terminal loops of helices 6 and 12 indicate a tertiary interaction unique to HeLa 18S rRNA. We have confirmed the existence of the complex tertiary folding recently proposed (Gutell and Woese 1990 Proc. Natl. Acad. Sci. 87, 663- 667) for the universally conserved helix 19 in HeLa 18S rRNA. The complementarity of chemical modifications and enzymatic cleavages provided experimental evidence for the proposal of a model structure for the 655 nucleotides of the 5' domain of HeLa 18S rRNA. INTRODUCTION Electron microscopic analysis of the small subunits of bacterial and eukaryotic cytoplasmic ribosomes has revealed considerable structural differences in their size and morphology (1). Varieties of other techniques have confirmed that the eukaryotic small subunits are larger and more complex than those of the prokaryotes (2,3). The small ribosomal subunits of prokaryotes *

To whom correspondence should be addressed

contain only 21 different proteins (4,5), while the eukaryotes contain up to 37 different proteins (6) which are also bigger in size. This increase in protein number also shifts the protein content of the small ribosomal subunit from about 40% in prokaryotes (7) to about 55% in mammals (8). The protein synthetic machinery in eukaryotes is further complicated by the enormous number of factors involved in the initiation of protein synthesis (9,10). However, the small ribosomal subunits of all organisms share a common feature by containing a single RNA molecule of 16- 18S. The 18S rRNAs of eukaryotes in general contain 300 more nucleotides than the prokaryotic counterparts and these are mostly inserted in two distinct regions in the 18S rRNA (11). Comparative sequence analysis of the rRNAs of the small ribosomal subunits resulted in proposal of generalized secondary structure models (12-15). The secondary structure model for Escherichia coli 16S rRNA has been investigated by chemical (16,17) and enzymatic (18,19) probing and has resulted in general agreement on its structure (20,21). In contrast, various secondary structural models have been proposed for eukaryotic 18S rRNA mostly due to the extensive divergence of sequences and lack of sufficient experimental data. The secondary structure models proposed for yeast (22-24), rabbit (25) and Xenopus (26) small subunit rRNAs were partially tested by chemical or enzymatic probing. In the present study, the 5' domain of human 18S rRNA derived from HeLa 40S ribosomal subunits was subjected to detailed structural analysis using both chemical and enzymatic probing techniques. -

MATERIALS AND METHODS Preparation of rRNA HeLa S3 cells were grown in MEM medium in suspension cultures. Ribosomes were prepared by a modification of the procedure (27). The frozen cells were suspended in the lysis buffer (10 mM Tris-HCl, pH 7.5 at 25°C, 10 mM KCI, 1 mM Mg(OAc)2, 10 mM fl-mercaptoethanol) and homogenized for a few min. The extracts were gently mixed with 1/10th volume of 3 M KCI and 20 mM Mg(OAc)2. The nuclei and mitochondria were removed by centrifugation at 25,000 g for 30 min. The post-mitochondrial supematant was first treated with

7056 Nucleic Acids Research, Vol. 18, No. 23 Brij 58 followed by sodium deoxycholate to a final concentration of 0.7% . Ribosomes were pelleted through a 60% sucrose (w/v) cushion prepared in 25 mM Tris-HCl, pH 7.5, 100 mM KCl, 10 mM Mg(OAc)2, 10 mM 3-mercaptoethanol at 120,000g for 16 h at 4°C in a Ti45 rotor. Ribosomes were dissociated into subunits in the presence of 1 mM puromycin (28). The ribosomal subunits were separated in 10-40% sucrose (w/w) gradients in a Zonal Til5 rotor at 27,000 rpm for 16 h (29). The subunits were recovered by centrifugation after increasing the Mg++ concentration to 10 mM. 18S rRNA was extracted from the 40S subunits by the phenol:chloroform method (30). The rRNA was precipitated by ethanol and purified further on 15 - 30% sucrose gradients (30). The purity of the rRNA was checked by agarose gel electrophoresis (31).

Chemical modifications of 18S rRNA The isolated 18S rRNA was renatured in 330 mM KCI, 20 mM Mg(OAc)2, and 1.0 mM DTT with either 80 mM Hepes-KOH (pH 7.5) or 80 mM borate buffer (pH 8.0) at 42°C for 1 h. Chemical modifications were carried out at 4°C as described (17,32). DMS, Kethoxal and DEPC modifications were in HepesKOH buffer, and CMCT modification was in borate buffer. The reactions were terminated by adding five-fold excess of E. coli 50S subunits (and also DMS stop buffer in the case of DMStreated sample) and 2.5 volumes of ethanol. The rRNA was extracted by phenol:chloroform (30).

Partial nuclease digestion Nuclease digestions were carried out in 80 mM Hepes-KOH, pH 7.5, 330 mM KC1, 20 mM Mg(OAc)2 (33). 25 jg of 18S rRNA (0.1 ml) was incubated with 0.25 units of cobra venom nuclease (Pharmacia) or 0.2 units of RNase TI (Pharmacia) at 4'C for 30 min. The reactions were terminated by the addition of 125 yg of E. coli 50S subunits followed by 0.1 ml of 2 x RNA extraction buffer (30). RNA was extracted by phenol:chloroform and precipitated by 2.5 volumes of ethanol. Primer extension The numbering of primers was based on the first nucleotide incorporated into cDNA. The following primers were used for the analysis of the 5' domain: (158)ATGTATTAGCTCTAGAAT, (222)CGGGTTGGTTTTGATCTG, (300)TGCGATCGGCCCGAGGTT, (368)GCACGGCGACTACCATCG, (426)CAGGCTCCCTCTCCGGAA, (508)TTATTTl?TCGTCACTACC, (569)TCGTTAAAGGATTTAAAG, (664)AACTACGAGCTTTTTAAC. Reverse transcriptase directed primer extension was carried out by a modification of procedures described earlier (17,32). One and one-half pmoles of 18S rRNA were mixed with 1 pmole of oligonucleotides and heated at 90'C for 1 min. Hybridization was at room temperature for 30 min. The rRNA was then mixed with 4 kd of a cocktail containing deoxynucleotides (110 zM each of dGTP, dCTP, dTTP and 25 AM dATP), reverse transcriptase (0.5 unit/Al), Mg+ +, DTT and [a!32P] dATP (400 Ci/m mol). The sequencing samples also contained 1 yd of ddTTP (100 MM), ddGTP (50 AM), ddCTP (25 AM) or ddATP (10 AM). cDNA synthesis was carried out at 42°C for 30 min. One 1l of cold deoxynucleotides (2 mM) was added and the reaction was continued for 15 min. The reaction was terminated by adding 6 Al of 98% formamide containing xylene cyanol and bromophenol blue. The samples were denatured at 90°C for 5 min and rapidly chilled. Three Al of each sample were loaded on a 10% polyacrylamide/urea gel.

Electrophoresis was performed at 60 W constant power for 3.5-4 h. The gels were fixed in 15% methanol and 7% acetic acid and dried.

RESULTS AND DISCUSSION A combination of chemical and enzymatic probing was employed to unambiguously identify the unpaired and paired bases in the HeLa 18S rRNA. The four chemical reagents used here differ in base specificities. DMS reacts with the unpaired adenine at Ni, cytosine at N3 and guanine at N7 positions (34), but methylation at N7 of guanine cannot be detected by the primer extension technique used here. RNase T 1 and kethoxal specifically react with unpaired guanines; kethoxal reaction with the NI and N2 of guanines results in the formation of an exocyclic ring (35). DEPC carbethoxylates adenines and guanines at the N7 position and this modification ultimately results in the opening of the imidazole ring between the N7-C8 bond. The reactivity of DEPC is sensitive to stacking interactions and adenines in the helices are nonreactive. CMCT modifies unpaired U and G at the N3 and N4 positions, respectively. CV nuclease, on the other hand, is not specific to any base but cleaves at helical (doublestranded) regions in the rRNA (36). The chemical and enzymatic reactions were carried out at 4°C because the chemical reactivity at this temperature did not interfere significantly with ribosome functions in E. coli (17). Modified bases were detected by primer extension using reverse transcriptase. This enzyme pauses at the site of modification resulting in the formation of intense bands one nucleotide below the modified base (17,32) and by correspondence to the dideoxy sequencing lanes the modified bases were precisely identified (Fig. lA & B). The human 18S rRNA primary structure derived from gene sequencing (37-39) contains 1868- 1870 nucleotides. Although there is overall agreement on the sequence data obtained from three different laboratories, there are some discrepancies too. The sequence information which fits very well with the cDNA sequences derived from this study was used as a reference for corrections wherever applicable. The rest of the sequence data is based on results reported by McCallum and Maden (38). In comparison to the generalized secondary structure for eubacterial 16S rRNA, nucleotides 1 -655 are grouped as the 5' domain of human 18S rRNA. Based on the system used by Dams et al. (15), the 5' domain is classified into 19 helices and these helices were interconnected by single stranded regions.

Helices 1-4 The 5' end of HeLa 18S rRNA is folded into the pseudoknot structures as proposed for E. coli 16S rRNA. The two pseudoknot structures at the 5'end of the 16S rRNA were postulated to be coaxial (40). Bases U9, GIO and All which lie as unpaired between helices 1 and 2 were reactive to single strand specific reagents (Fig. 2A). In helix 2, the paired AGGA (1196- 1199) were reactive to single strand specific reagents (data not shown) and these modifications are not in conformity with the model. This part of helix 2 is also modified by single strand specific reagents in E. coli (17), rabbit (25) and yeast (22) rRNAs. The modifications observed in E. coli 16S rRNA were attributed to the unusual pseudoknot structure present in helix 2 (17). Though cleavage of A22 and C30 by CV nuclease supports their paired existence in helix 3, the reactivity of the paired G646, U647 and A648 to single strand specific reagents were in disagreement with their pairing pattern. Comparative sequence analysis indicated the conservation of the C-A pair in eukaryotic and mitochondrial

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Nucleic Acids Research, Vol. 18, No. 23 7057

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Fig. 1 A and B. Chemical and enzymatic probing of HeLa 18S rRNA by primer extension. 18S rRNA isolated from HeLa 40S subunits was modified by DMS (Lane 1), Kethoxal (lane 2), DEPC (lane 3), CMCT (lane 4), CV nuclease (lane 5) or RNase Ti (lane 6). The modified bases were identified by primer extension using synthetic deoxyoligonucleotides and reverse transcriptase. A, C, G, and U were dideoxy sequencing lanes and correspond to the sequences that appear in the rRNA. 0 is the unmodified 18S rRNA control to identify nonspecific termination in the primer extension reactions.

rRNA (12). Though the protection of C24 is suggestive of its involvement in base pairing, A650 is methylated at Ni indicating that the C-A pairing could be an unusual pairing through the N6 and N7 of adenine. Two different models have been proposed for the folding of helix 4 (15,41). In De Wachter's model (15), the helix contains four base pairs while in Noller's model (41) it is formed by six base pairs. Though one of the strands is common, the complementary strand is 515-518 (15) or 562-569 (41). Since the reactivity of bases 562-564 to single strand specific reagents disagrees with Noller's pairing pattern but is in agreement with De Wachter's proposal, the pairing pattern of the latter model was used in Fig. 2 A & B. Furthermore, in two eukaryotic small subunit rRNAs (42,43) helix 4 cannot be folded into a model as proposed by Noller, but folding those sequences into a structure similar to the one shown in Fig. 2 A&B is feasible (14). The CV nuclease cleavages at U36 and C37 suggest that the bases are at least stacked because CV nuclease also cleaves stacked bases (44). The stretch of 11 unpaired nucleotides (36-46) which interconnect the helices 4 and 5 was confirmed as all of the bases except U36 and C37 reacted with single strand specific reagents (Fig. 2A).

Helices 5-7 C48 and C49 were modified at N3 and were also CV nuclease sensitive, indicating that these modifications by DMS might have occurred in the double stranded region. The cleavage at C479 by CV nuclease supports its pairing with C47. The bulged A50 was not reactive to either enzymatic or chemical probing. Similarly, the bulged A in this helix was protected from Ni and N7 modifications in rabbit 18S rRNA (25). The single stranded nature of the 8 nucleotides (54-61) which join helices 5 and

6 was in reasonable agreement with the chemical and enzymatic probing data. However, U57, C58 and U59 which exist as unpaired were cleaved by CV nuclease (Fig. lA) implying their involvement in base pairing probably in tertiary interactions. Base A54 was reactive with DMS, DEPC and CV nuclease supporting the stacked nature of this base. Of the 9 nucleotides (66-74) in the terminal loop of helix 6 only A68 was methylated. A68, G71 and C72 of this loop were cleaved by CV nuclease which supports their involvement in base pairing probably in tertiary interactions (see section on helix 12). G62 (paired to C78) and U63 (paired to A77) were reactive to single strand specific reagents, while A77 and C78 were cleaved by CV nuclease. The reactivity patterns indicate that this helix may be folded uniquely in different organisms. Support for this conclusion stems from comparative sequence analysis which indicates that this helix is very irregular in addition to its extremely variable secondary structure and size (13). While the corresponding helix was completely eliminated in mammalian mitochondria, plant mitochondria have inserted some 120 nucleotides in this region (12). The existence of the long stretch of 17 unpaired nucleotides (79-95) which interconnect helices 6 and 7 agrees with the model as 12 of these bases were reactive to single strand specific reagents (Fig. 1A). However, four nucleotides (86-89) located in the middle of these unpaired bases were not reactive to single strand specific reagents. The cleavage of C86, U87 and G88 by CV nuclease (and also kethoxal reactive G90) suggests their involvement in base pairing most likely in tertiary interactions. The formation of helix 7 by the pairing of bases 96-98/432-434 was confirmed as U97 was cleaved by CV nuclease (Fig. 2B). Kethoxal reactivity at G432 could be explained by the unusual nature of the helix, due to the joining

7058 Nucleic Acids Research, Vol. 18, No. 23

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Fig. 2. The derived secondary structure model of HeLa 18S rRNA. The reactivity of bases in 18S rRNA to single strand specific chemical reagents is presented in Fig 2A. The cleavage at helical regions by CV nuclease is presented in Fig 2B. The three sizes of circles denote the degree of reactivity from low, medium and high, respectively . Every fiftieth nucleotide is numbered and the tenth nucleotide is marked by a line. The helix numbers are given in bold italics.

of helices 7 and 13. The unpaired nature of nucleotides 99-105 was in agreement with the proposed models as 6 of the 7 bases were reactive to single strand specific reagents. U105 (modified to pseudouridine, ref. 45) and G407, though unpaired, reacted only with CV nuclease indicating the possibility of a U-G pair. Teriary interactions between the U105 and A408 were postulated for bacterial 16S rRNA (13) and UV crosslinks were also identified between these regions in 30S subunits (46). Helices 8-9 Reactivity of U109, Ul10, A111 and U112 with single strand specific reagents were well in agreement with the proposed model. Strong terminations occurred in between nucleotides 110-125 in cDNA synthesis (Fig. IA). Using ca [35S] dATP (data not shown) the pausing of reverse transcriptase was mapped to U1 16 and U121. These nucleotides are 2' 0-methylated in HeLa 18S rRNA (45). A349 modified at NI, was also cleaved by CV nuclease indicating that the base may be paired. In the 342-346 loop all bases except C342 were reactive, while in the opposite loop (118-121) only C1 18 was reactive to single strand specific reagents . The reactivity of C337, C340 and C350 to CV nuclease supports their paired nature. Furthermore, the chemical modifications at G351, U352 and G355 led helix 8 to

the pairing pattern as shown in Fig 2A & B. This pairing scheme is in agreement with De Wachter's model (15). Comparative sequence analysis has predicted that helix 8 is completely irregular and it has been eliminated from mammalian mitochondrial rRNA (12). The existence of 15 unpaired bases (127-141) between helices 8 and 9 could not be fillly confirmed since only 5 of these bases were reactive to single strand specific reagents. Cleavage of nucleotides 142-148 and the complementary 175 and 176 with CV nuclease supports the pairing pattern indicated in Fig 2B. The highly conserved A149 and A150 were modified at NI and N7 confirming their unpaired nature. In the terminal loop (157-161) of helix 9, four of the five bases reacted to single strand specific reagents. Strong reactivity was also observed in the corresponding loop of E. coli 16S rRNA (17). The bulged G153 and G165 were not reactive to single strand specific reagents and may be involved in G-G pairing as proposed from phylogenetic comparison (12). However, this G-G pair is insensitive to CV nuclease and its existence can only be inferred. Since NI and N2 of these two guanines were nonreactive, the possible bonding could be through the N3 and N2 (37). The reactivity of A170 to DMS, DEPC and CV nuclease indicates that this base may be stacked. Modification of universally conserved C151 of C151-G168 pair and the paired U152 by single

Nucleic Acids Research, Vol. 18, No. 23 7059

strand specific reagents was not in conformity with either of the models. There is a strong possibility of a U-U pair between 143 and 176, as both these bases were cleaved by CV nuclease and protected from chemical modifications. G-A pair between G146 and A173 is possible from CV nuclease reactivity at G146. Since the NI of G and A were protected and A173 is modified at N7, the possible configuration of bases in this pair could be G(trans)A(trans) in order to facilitate such a pairing (17). Helices 10-10.2 Helix 10 exists only in the model proposed by Dams et al.(15), while the regions remain unpaired in Noller's model (41). The absence of chemical reactivity within these bases (Fig. 1A) is consistent with the existence of helix 10. Of the four unpaired bases which interconnect the helix 9 to 10 two bases (G180 and A181) were reactive to single strand specific reagents. G308, G309, G312 and A313 which remain unpaired between helices 10 and 11 were reactive to single strand specific reagents (Fig. 2A). In the terminal loop of eukaryotic specific helix 10.1, bases 198-201 were modified by single strand specific reagents (Fig. 2A). A191 and A209 which exist as unpaired in Noller's (41) and De Wachter's (15) models were reactive only at NI position, and not at N7, indicating stacking. It is possible that G190/A209 and A191/G208 are involved in G-A and A-G pairings. In the A-G pairs two hydrogen bonds are made, (A)N6H... 06(G) and (A)N7...HNI(G). This kind of hydrogen bonding would result in the modification of A at NI and protection of G at NI. The pattern of chemical reactivity of these bases in HeLa 18S rRNA is fully consistent with A(syn) -G(trans) pairing as proposed by Traub and Sussmann (47). However this proposal cannot be fully confirmed due to the absence of any CV nuclease cleavage at these sites in the partial nuclease digestion method used here. It has been proposed that the A-G pair is involved in joining two helices whose axes are almost, but not quite, coaxial (12) and might function in a similar way in helix 10.1 of HeLa 18S rRNA. The nonreactivity of U189 and U210 suggests the existence of a noncanonical U-U pair in this helix. Recent studies indicate that U-U pairing is much more frequent in eubacterial 16S rRNA than hitherto suspected and such noncanonical pairing is more favored in the middle of the helix (48). These pairs have increased the length of the helix 10.1 in HeLa 18S rRNA by three base pairs. It is reasonable to believe that the observed changes in the primary structure of HeLa 18S rRNA, in comparison with those of other eukaryotes, were for enhancing the helical content of the rRNA. Of the 5 unpaired nucleotides (214-218) which span between the helix 10.1 and 10.2, two bases (G215, A217) were modified by single strand specific reagents. CV nuclease cleavage at U222, C223, C228, A229, A230, C25 1, U252, U285 and U286 is in support of their paired nature in helix 10.2, as in the model proposed by Noller (41). In the terminal loop of eukaryotic specific helix 10.2, C259 and C260 were highly reactive at N3. In the bulged loop of four nucleotides (281-284) no bases reacted with single strand specific reagents, but C284 was cleaved by CV nuclease indicating base pairing. In the complementary strand, the ACC (232-234) sequences were reactive to CV nuclease suggesting the possibility of an A-C pair and two C-G pairs as presented in Fig. 2A & B. There is no phylogenetic evidence to support the presence of an A-C pair in this helix in eukaryotic 18S rRNA. But the mammalian mitochondrial rRNA sequences showed remarkable number of A-C pairing in various helices (13). Here again such a pairing

enhanced the length of the helix. Contrary to their paired nature, A301 and A302 were modified at NI and N7. The reactivity of G225, A235, A236, C237, A244, C271 and A292 to single strand specific reagents was in agreement with the model. All bases except A292 of the GACUCUA (291-297) sequences were nonreactive to single strand specific reagents. Helices 11-12 In helix 11, the pattern of folding as suggested by Dams et al. (15) was in agreement with the chemical modification data (Fig. 2A). A318 was reactive to DMS and DEPC supporting its bulged nature. The sensitivity of C321 and C322 to CV nuclease and the reactivity of C323 -326 and U328 to single strand specific reagents (Fig. iB) supports the pairing pattern as shown in Fig. 2A & B. Comparative sequence analysis supports the pairing between 356-358/402-404 in helix 12. In De Wachter's model (15) these bases (356-358) were unpaired. The CV nuclease cleavage at these sites (356 -358) favors the Noller's pairing pattern (41). Though the helix at this region is more extended in eubacteria, the phylogenetic data is less supportive for the pairing in eukaryotes (48). In the terminal loop of helix 12 (378 -386) only U378, C379, and C386 reacted with single strand specific reagents. The GCCGUG (380-385) sequences of the loop was not reactive to single strand specific reagents but was reactive to CV nuclease (380-383) implicating paired nature of these sequences. Similarly, in helix 6 the unpaired CACGGC (67-82) were also protected from single strand specific reagents but reactive to CV nuclease (A68, G71 and C72). It is possible that there were tertiary interactions between helices 6 and 12 through the terminal unpaired nucleotides in HeLa 18S rRNA. In E. coli 16S rRNA most of the bases in the terminal loop of helices 6 and 12 were modified by single strand specific reagents (17) and such interactions between helices 6 and 12 may not be favored. In the 3D model proposed for E. coli 30S subunits based on protein -RNA and RNA -RNA crosslinks, helices 6 and 12 are located very close to one another (49). Based on the structural analogy between 16S and 18S rRNA, hydrogen bondings between the terminal nucleotides in helices 6 and 12 are probable in HeLa 18S rRNA. Furthermore, the reduced size of helix 6 in HeLa 18S rRNA (13) may be consistent with a coaxial helix between helices 6 and 12. However, it remains to be seen whether these two loops can be crosslinked and if such a pairing exists in the 40S subunits. In the bulged loop of helix 12 (359-364), all bases except U359 showed reactivity to single strand specific reagents, but U359, U361 and U362 were cleaved by CV nuclease (Fig. 2B). The unpaired U367, U368, C369 and G370 and the paired C365, U366 and A371 were reactive to single strand specific reagents (Fig. 2A) in helix 12. Bases U375, C390, C391 and A392 were reactive to CV nuclease supporting their paired nature. The stretch of six unpaired bases (397-402) was confirmed by their reactivity to single strand specific reagents. Obviously there are considerable differences in the chemical modifications of helix 12 between E. coli and HeLa. The unpaired A408 was only reactive at N7 position and may be involved in hydrogen bonding. Helices 13-15 The paired nature of G412 (paired with U424) and G413 (paired with C428) in helix 13 was confirmed by CV nuclease cleavage. In the terminal loop, however, A 418 was reactive to DEPC alone and none of the other bases were sensitive to single strand specific probes. The results of chemical modifications in helix 13 of HeLa

7060 Nucleic Acids Research, Vol. 18, No. 23 18S rRNA are very different from those obtained in E. coli 16S rRNA where all of the bases in the terminal loop are modified by single strand specific reagents (17). The strong reactivity of A414 and the mild reactivity of A415 at NI alone in 18S rRNA may be because of the noncanonical pairings through the N7, N6 of adenines with N3 and 02 of Us (37). The reactivity of paired G431 with kethoxal may be due to unusual helix formation at this region. The bulged GAU (425-427) bases were not reactive to single strand specific reagents. The base corresponding to A426 was crosslinkable to helix 3 in E. coli 30 subunits (21). It is reasonable to believe that such tertiary interactions were conserved in HeLa 18S rRNA and the absence of chemical reactivity between 425 -427 could be due to the tertiary pairing. The helix 13 in E. coli assumes two different conformations in the native 30 S subunits and the free 16S rRNA (17). Ribosomal protein S16 binds to this helix and the association of S16 shifts the helix from one conformation to another in E. coli 16 S rRNA (50). Whether such a mechanism exists in HeLa ribosomes remains to be established. One of the characteristic features identified by phylogenetic comparison in helix 14 of all organisms is the conservation of a G-A pair though not at the same exact location (12). The protection of G438 and A455 from chemical reagents was more in agreement with the existence of the G-A pair in HeLa 18S rRNA probably in G(trans)-A(trans) configuration. C441 and C442 paired with G452 and G451 respectively, were cleaved by CV nuclease in agreement with their paired nature. A CV nuclease cleavage site has been observed in helix 14 in E. coli 30S subunits and this site is protected in 70S ribosomes (51). In the highly conserved terminal loop of this helix (443 -450), six of the eight nucleotides were reactive with single strand specific reagents. The reactivity pattern in HeLa 18S rRNA is almost identical to that observed in E. coli 16S rRNA (17). Helix 15 is a very short and consists of four base pairs in all organisms. The bulged A460 was highly reactive at N7 position. The complementary A469 was not modified indicating the possibility of an A-A pair formation. Comparative sequence analysis has predicted the existence of an A-A pair in eukaryotes except for D. discoideum (12). In the terminal loop of helix 15 (463 -466), none of the bases were reactive to single strand specific reagents or to CV nuclease. In E. coli 16S rRNA, two of the four bases in the terminal loop were reactive to single strand specific reagents (17). In the highly conserved GCA (471 -473) bases which connect helices 15 and 5 only C472 was reactive with DMS but the other two nucleotides were protected. In E. coli 16S rRNA, the bases corresponding to 471 and 473 were reactive while C472 was protected (17). Bases 483 -485, which span between helices 5 and 16, were reactive to single strand specific reagents but the other three nucleotides (480-482) though unpaired in the model were protected (Fig. 2A & B). Although these bases were highly reactive to chemical modifications in E. coli 16S rRNA, they are involved in tertiary interactions with helix 4, since UV crosslinks were identified between these regions in 30S subunits (21). It is possible that the protection found in 18S rRNA between 480-482 is due to tertiary interactions similar to those identified in 30S subunits. Helices 16-18 In the terminal loop (499-503) of helix 16, G499 and A500 were modified by single strand specific reagents. Nucleotides 501-503 were protected from both single strand specific and double strand specific probing methods. This reactivity pattern is very different

from that observed in E. coli 16S rRNA (17). The paired C498/G504 were reactive to single strand specific reagents probably due to their close proximity to the terminal loop region. In the conserved sequence present in the bulged interior loop (492 -494), all the bases were reactive to single strand specific reagents. Unlike in E. coli 16S rRNA, the bulged A508 was protected from chemical modifications. The modification of A486 could be due to its proximity to the loop region. Paired U487,U488, A489 and their complementary G513, A512, and U511 were reactive to single strand specific reagents (Fig. 1B) contrary to their pairing pattern. Comparative sequence analysis has shown extensive divergence in the organization of helices 17 and 18 in eukaryotes and eubacteria (12). Crosslinking (21) and chemical protection studies (52) have shown that these two helices are the sites of interaction for the 30S subunit assembly inducing protein S4. The paired 532-535, C550 and U556 of helix 17 were reactive to CV nuclease supporting the pairing pattern shown in Fig. 2A&B. Reactivity of A536 at Nl and also by CV nuclease supports the possibility of an A-G pair with G547. The protection of G547 from NI modification is fully consistent with the A(syn)-G(trans) pairing as described earlier (47). The C548 was unpaired as concluded from its modifications at N3. In the terminal loop of helix 17, U540, U541, U542 and C543 were reactive to single strand specific reagents confirming their unpaired nature. The bulged A554 and the paired A555 were reactive to N7 modification by DEPC indicating that they are not stacked. The unpaired nature of bases 519-526 preceding the helix 17 was confirmed as all of the bases were reactive to single strand specific reagents. Helix 18 is folded in the format shown in Fig. 2A & B based mainly on the reactivity of bases to chemical modifications, and it is completely different from the pairing pattern proposed by Dams et al. (15). In the unpaired nucleotides which were found at the stalk of helix 18, only C570 and A575 were reactive to single strand specific reagents. Cleavage of bases 578-581 by CV nuclease is supporting their paired nature. In the terminal loop of helix 18, three of the four nucleotides were reactive to single strand specific reagents. Reactivity of C567, C568 and A569 to CV nuclease implies their paired nature. In Noller's model (41) these were part of the nucleotides that are involved in base pairing in helix 4. Because of the reactivity of bases 562-564 to single strand specific reagents we have represented all the bases (559-575) as unpaired. The protection of many of the other bases to chemical and enzymatic probing could not be well explained.

Helix 19 This is one of the most highly conserved regions in the small ribosomal subunit RNA in all organisms and exists as a compound helix (13). There is strong evidence from site-directed mutagenesis (53,54) and from chemical protection studies (55 -57) in E. coli ribosomes that this helix is actively involved in ribosome functions. There are at least two different pairing patterns proposed for this helix (15,41). The major difference between these two models are in the bulged residues and in the terminal loops (15,41). Though A599 was paired, it was reactive at N 1 and N7 position probably due to its proximity to the loop region. Bases A604 and A605 were reactive at Nl and N7 while A604 was also cleaved by CV nuclease which is indicative of stacking. CV nuclease cleavage at C608, U609, C614 and C615 was more in support of the pairing pattern proposed by Dams

Nucleic Acids Research, Vol. 18, No. 23 7061 et al.(l5). In eubacterial 16S rRNA, the terminal loop of helix 19 was postulated to be involved in tertiary interaction with the bulged loop at the base of the helix (58). The pairing pattern proposed for eubacterial 16S rRNA was between 620-622/601-603. In HeLa 18S rRNA, G620 was reactive at N1 and was also cleaved by RNase Tl (Fig. iB). The G620 reactivity to RNase Tl and kethoxal may be because it is the terminal pair of the tertiary helix and the adjacent A619 is unpaired. The corresponding base in E. coli 16S rRNA was also modified by kethoxal (17). The reactivity of C622 to CV nuclease in HeLa 18S rRNA is in agreement with the proposed tertiary pairing. It has been argued that the proposed canonical pairing in the tertiary interactions may be inconsistent in the eukaryotic small subunit rRNAs (58). However, the results presented on HeLa 18S rRNA (Fig. 2A & B) are fully consistent with the tertiary pairing proposed for the eubacterial rRNA. Based on the sequence data from Chlamydomonas reinhardtii, it has been recently postulated that one strand of this pseudoknoted tertiary helix is enclosed by another helix formed by the pairing of 617 -618/623 -624 (48). The absence of chemical reactivity of 617 -618 and CV nuclease cleavage at 623 and 624 fully support such an interaction also in human 18S rRNA. The CV nuclease reactivity of bases 630-634 favors their paired nature. The paired A640 was modified at NI position but was also cleaved by CV nuclease indicating that the DMS modification might have occurred in the helical region. Cleavage of U630 and U631 by CV nuclease, though they are unpaired in the model, may be due to their involvement in tertiary interactions. There are differences between the chemical reactivity patterns reported here and the adenine modifications observed in rabbit 18S rRNA (25). One of the causes for this discrepancy can be attributed to the reaction temperature used for the chemical modifications. We have performed the chemical modifications at 4°C, while the rabbit 18S rRNA modifications were carried out at 37°C (25). It has been pointed out that chemical modifications at 37'C inactivate the ribosomal particles while the modifications at 4°C affect the ribosome functions only marginally (17). Furthermore, the low temperature may be stabilizing many weak structural interactions (17). One other parameter which might have influenced these two results may be the salt conditions used. It has been previously shown that an increase in salt content enhances the folding of rRNAs (59-61). Consequently we found only very little modification within the helix in the 5' domain of HeLa 18S rRNA. The 5' domain of small subunit rRNA is the site of initiation of the assembly process in E. coli and this initiation process is directed by the interaction of ribosomal protein S4 (62,63). Nucleotides between 60-300 in E. coli 16S rRNA were resistant to chemical modifications (17) and these protections were attributed to the formation of a core structure for the assembly of 30S subunits (64). In HeLa 18S rRNA, we were unable to identify such a specific region resistant to chemical modifications at the 5' domain. This suggests that the assembly of the 40S subunit does not progress through the formation of a core structure similar to that proposed for E. coli 30S subunits. It is possible that such a core structure might have been provided for by transcribed spacers in eukaryotes for the assembly of ribosomes. This may explain the difficulties encountered in reconstituting mature 18S rRNA into functional 40S subunits in vitro. Furthermore, the extensive protection from chemical modifications observed in the terminal loops of many helices is indicative of unique tertiary interactions occuring in eukaryotic 18S rRNA.

CONCLUSIONS By chemical and enzymatic probing and by comparison with phylogenetic data, we have derived a secondary structure model for the 5' domain of the HeLa 18S rRNA. One of the interesting observations from the analysis reported here is the confirmation of the existence of a pseudoknotted tertiary folding of the universally conserved helix 19, in agreement with a previously proposed structure (58). The methods employed here have also led to the identification of certain noncanonical pairing in some of the helices in the 5' domain of HeLa 18S rRNA. Of the several noncanonical pairing proposed for 18S rRNA only U-U pair (helix 9), G-A pair (helix 9), A-C pair (helix 10.2) and A-G pair (helix 17) were confirmed from the CV nuclease cleavages, while the other C-A (helix 3), G-G (helix 9), A-G (helix 10.1), G-A (helix 10.1), A-A (helix 15) pairs can only be inferred by their protection from chemical modifications. However, based on the selective reactivity of bases to chemical reagents, the possible configuration of the bases involved in the noncanonical pairing can be predicted.The bases in many of the terminal loops at the 5' domain were resistant to chemical modifications, and this is often in complete disagreement with the reactivity observed in E. coli 16S rRNA. This observation indicates the existence of unique tertiary interactions in HeLa 18S rRNA through the terminal loops. One such interaction may be between helices 6 and 12 of HeLa 18S rRNA. There is good agreement with the chemical modification data and the predicted secondary structure from the comparative sequence analysis in many of the helices of the 5' domain. However, the pairing of helix 18 is exclusively based on chemical modification data. This pairing has resulted in grouping a large number of nucleotides (559-575) as unpaired though there were CV nuclease sensitive sites within these bases. The organization of this region cannot be considered as absolute because of its extensive divergence in its primary structure. The analysis of the secondary structure of the central and 3' domains is in progress and these studies may shed more light on the tertiary interactions involved in the folding of the eukaryotic 18S rRNA.

ACKNOWLEDGEMENTS We thank Drs. Santa J. Tumminia and Carl Weitzmann for discussions and careful reading of the manuscript.

ABBREVIATIONS USED DMS = dimethylsulfate; Kethoxal = f ethoxy-aketobutyraldehyde; DEPC = diethylpyrocarbonate; CMCT = 1 cyclohexyl-3-(2-Morpholinoethyl)carbodiimide metho-p-toluene sulfonate; CV nuclease = cobra venom nuclease.

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